Measurement of reactive oxygen species in brain and heart tissue

Measurements of ROS generation as hydrogen peroxide (and converted superoxide) was measured using the O2k-Fluorescence LED2-Module attached to the Oroboros O2k-FluoRespirometer, permitting simultaneous measurements of hydrogen peroxide (H₂O₂) production and mitochondrial respiration, utilizing an Amplex UltraRed assay as previously described [9]. In short, Amplex UltraRed (N-acetyl-3, 7 dihydroxyphenoxazine) (5 mM), in the presence of horseradish peroxidase (1 U/ml), reacts with H₂O₂ to produce the fluorescent compound resorufin. The addition of superoxide dismutase (SOD) (10 U/ml) ensures that all superoxide is converted into H₂O₂. A 3-point calibration of the fluorometric signal was done prior to each measurement by the addition of 100 nM H₂O₂. Mitochondrial ROS generation is the predominant source of ROS and leads to alterations in redox signaling, oxidative damage to proteins and lipids, additional mitochondrial dysfunction and ultimately a major cause of ongoing secondary brain and heart injury.